In vivo the neutral proteinase collagenase is a prime mediator of joint destruction in rheumatoid arthritis, and in vitro cultures of rheumatoid synovial cells "spontaneously" secrete large amounts of this enzyme into culture medium. Mechanisms governing either induction or prevention of its synthesis are not known, and to date only corticosteriods decrease collagenase synthesis under physiologic conditions. Cultures of rabbit synovial fibroblasts provide a good model for studies on induction of collagenase synthesis. Untreated cultures secrete negligible amounts of collagenase, but treatment with the tumor promoter phorbol myristate acetate results in production of significant quantities of this enzyme. It is secreted in latent form and appears in culture medium only after a 24-hour lag. Concomitant treatment of cells with alpha-amanitin (2 micrograms/ml), an inhibitor of RNA polymerase II, prevents synthesis of collagenase, thus suggesting that induction of enzyme is due to induction of a new messenger RNA. We plan to pursue this hypothesis by isolating the m-RNA for collagenase from cells treated with phorbol myristate acetate, to translate the message in an in vitro rabbit reticulocyte lysate system, and to detect the translation product by radioimmunoassay and by assays measuring functional collagenolytic activity. Then by enriching rRNA populations for collagenase mRNA and by using this mRNA as a cDNA probe, we plan to isolate the gene for collagenase from rabbit cellular DNA and to amplify it by insertion into a bacterial plasmid. These studies support our ultimate goal of understanding mechanisms regulating collagenase production in both normal and disease states.